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A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain
[Image: see text] Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10178785/ https://www.ncbi.nlm.nih.gov/pubmed/37126856 http://dx.doi.org/10.1021/acschembio.3c00092 |
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author | Anmangandla, Ananya Jana, Sadhan Peng, Kewen Wallace, Shamar D. Bagde, Saket R. Drown, Bryon S. Xu, Jiashu Hergenrother, Paul J. Fromme, J. Christopher Lin, Hening |
author_facet | Anmangandla, Ananya Jana, Sadhan Peng, Kewen Wallace, Shamar D. Bagde, Saket R. Drown, Bryon S. Xu, Jiashu Hergenrother, Paul J. Fromme, J. Christopher Lin, Hening |
author_sort | Anmangandla, Ananya |
collection | PubMed |
description | [Image: see text] Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC(50) 6.4 μM) and GS441524 (IC(50) 15.2 μM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC(50) 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC(50) 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors. |
format | Online Article Text |
id | pubmed-10178785 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-101787852023-05-15 A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain Anmangandla, Ananya Jana, Sadhan Peng, Kewen Wallace, Shamar D. Bagde, Saket R. Drown, Bryon S. Xu, Jiashu Hergenrother, Paul J. Fromme, J. Christopher Lin, Hening ACS Chem Biol [Image: see text] Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC(50) 6.4 μM) and GS441524 (IC(50) 15.2 μM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC(50) 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC(50) 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors. American Chemical Society 2023-05-01 /pmc/articles/PMC10178785/ /pubmed/37126856 http://dx.doi.org/10.1021/acschembio.3c00092 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Anmangandla, Ananya Jana, Sadhan Peng, Kewen Wallace, Shamar D. Bagde, Saket R. Drown, Bryon S. Xu, Jiashu Hergenrother, Paul J. Fromme, J. Christopher Lin, Hening A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain |
title | A Fluorescence
Polarization Assay for Macrodomains
Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2
Macrodomain |
title_full | A Fluorescence
Polarization Assay for Macrodomains
Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2
Macrodomain |
title_fullStr | A Fluorescence
Polarization Assay for Macrodomains
Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2
Macrodomain |
title_full_unstemmed | A Fluorescence
Polarization Assay for Macrodomains
Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2
Macrodomain |
title_short | A Fluorescence
Polarization Assay for Macrodomains
Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2
Macrodomain |
title_sort | fluorescence
polarization assay for macrodomains
facilitates the identification of potent inhibitors of the sars-cov-2
macrodomain |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10178785/ https://www.ncbi.nlm.nih.gov/pubmed/37126856 http://dx.doi.org/10.1021/acschembio.3c00092 |
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